Osseous tissue constitutes cartilage and the skeletal system, and has a mechanical function as a support and a site of muscle attachment, the functions of protecting vital organs and bone marrow, and the function of reserving ions, especially calcium and phosphate, for the maintenance of their homeostasis. Osseous tissue that functions as such is made up of a cell matrix such as collagen and proteoglycan, and various cells such as osteoblasts, osteoclasts, and osteocytes. Among them, osteoclasts are derived from hematopoietic stem cells and are responsible for the resorption of aged bone, and osteoblasts are derived from bone marrow stromal cells and are responsible for bone formation.
The murine monocytic cell line, RAW264.7 differentiates into multinucleated osteoclasts upon exposure to RANKL (receptor activator of nuclear factor κB (RANK) ligand). This differentiation is mediated by the binding of RANKL to the extracellular RANK domain to promote the activation of mitogen-activated protein kinase (MAPK), and in turn the translocation of transcription factor NF-κB into the nucleus to increase expression of osteoclast differentiation-related TRAP (tartrate-resistant acid phosphatase), MMP-9 (matrix metalloproteinase-9), and c-Src tyrosine kinase. The multinucleated osteoclasts formed during this differentiation function to resorb mineralized bone. In addition, the binding of RANKL to RANK promotes the activity of TRAF6 (tumor necrosis factor receptor-associated factor 6), leading to the activation of MAPK or transcription factors such as NF-κB, AP-1, and NFATc1 (Lee Z H, Kim H H. Signal transduction by receptor activator of nuclear factor kappa B in osteoclasts. Biochem Biophys Res Commun. 2003 May 30, 305, 211-4). Therefore, inhibition of the RANK/RANKL signal transduction pathway has been suggested as a promising approach for the treatment of bone diseases including osteoporosis.
Meanwhile, osteoblast cells are derived from mesenchymal stem cells, and mineralization such as calcium formation by osteoblast differentiation maintains bone integrity, which has an important role in the calcium and hormone homeostasis of the body. Calcium formation by osteoblast differentiation is controlled by vitamin D, parathyroid hormone or the like, and bone formation by the osteoblast differentiation is accomplished by synthesizing alkaline phosphatase (ALP) associated with osteoblast differentiation in the early stage by cross-talking between various signal transducers such as bone morphogenetic protein (BMP), Wnt, MAP kinase, calcineurin-calmodulin kinase, NF-κB and AP-I in the cell, followed by synthesizing mineralization-related elements such as osteopontin, osteocalcin, and type I collagen (Pittenger, M. F.; Mackay, A. M.; Beck, S. C.; Jaiswal, R. K.; Douglas, R.; Mosca, J. D.; Moorman, M. A., Simmonetti, D. W.; Craig, S.; Marshak, D. R. Multilineage Potential of Adult Human Mesenchymal Stem Cells. Science 1999, 284, 143-147). Therefore, since compounds stimulating alkaline phosphatase activity promote osteocyte differentiation, they can be developed as a therapeutic agent for bone diseases.
The growth of bone is controlled by bone remodeling which maintains a balance between the actions of bone resorption by osteoclast and bone formation by osteoblast. However, over-activation of the osteoclast or reduced activation of the osteoblast causes an imbalance in the bone remodeling cycle to disrupt the balance between osteoclasts and osteoblasts in the body, leading to bone diseases.
A representative bone disease, osteoporosis occurs when the balance between bone formation and bone resorption is disrupted, and thus bone resorption occurs at a higher rate, leading to loss of bone density. In the United States, nearly 10 million people already have osteoporosis. Another 18 million people have low bone mass that places them at an increased risk for developing osteoporosis. One in two women and one in eight men are predicted to have an osteoporosis-related fracture in their lifetime, and about 2 million men in the United States already have osteoporosis. The annual direct healthcare costs for osteoporosis-related disease and bone fractures are estimated to be 14 billion dollars in the United States. In Korea, approximately 4 million people either already have osteoporosis or are at a risk for this disease, and the numbers are expected to skyrocket with today's aging population. This will impose enormous social, psychological, and economic burdens on individuals.
For the treatment of bone disease, the balance between osteoclasts and osteoblasts should be controlled, and current drugs to this end include bone resorption inhibitors and bone formation stimulators. Of them, bone formation stimulators have been actively studied, but enhancement of bone density by bone formation stimulators does not always lead to reduced bone fracture. Further studies are still needed regarding its clinical applications. Moreover, a drug for stimulating bone formation should have a low toxicity and be administered orally, because stimulators for activating osteoblasts and inhibitors for inhibiting bone resorption of osteoclasts should be administered to patients for a long period of time. Accordingly, there is an urgent need to study the drug.